We have recently demonstrated that the genomes of representative single-stranded positive-sense RNA (ss(+) RNA) viruses are decorated with a large number of post-transcriptional modifications (PTMs) and that the epitranscriptomic profiles of host cells undergo significant variations upon infection. At the same time, other groups have reported that N6-methyladenosine (m6A) ?one of these epitranscriptomic marks? affects nuclear export of HIV-1 RNA, packaging of hepatitis C viral RNA, and Zika virus gene expression. We therefore hypothesize that the numerous PTMs observed on these viral genomes may constitute the basis for pervasive regulatory mechanisms mediating viral gene expression and virus-host interactions. In preliminary studies, we have found that the dimethyl cytosine variants m5Cm and m44C were consistently present on the genomes of ss(+) RNA viruses and on cellular RNAs but were absent from the RNA of mock-infected cells. In this proposal we will use hepatitis C virus (HCV) as a model and m5Cm/m44C as a case-study to investigate the functions of PTMs in ss(+) RNA viruses, dissect the implications of PTMs in virus-host interactions, and elucidate the effects on viral and cellular gene expression. More specifically, we will apply antisense affinity capture and mass spectrometry (MS) analysis to identify the sequence position of m5Cm/m44C and other PTMs on HCV RNA genome. This information will guide a reverse genetics approach aimed at evaluating the function of the PTM at the selected modification sites. Next, we will perform depletion/overexpression of putative dimethyl cytosine writer enzyme to determine the effects on HCV translation, RNA replication/turnover, and virion assembly. Last, we will identify the cellular RNAs that are modulated by m5Cm/m44C by performing RNA-seq analysis of mRNAs modulated by dimethyl cytosine writer enzyme, isolating selected targets by antisense affinity capture, and submitting the captured material to PTM analysis. These data will enable us to evaluate the impact of mRNAs modulated by dimethyl cytosine and the dimethyl cytosine writer enzyme on HCV gene expression. This project will transform our view of the functions of viral RNA from mere templates for translation and replication to pivotal regulators of the infectious cycle. Beyond the virology field, the knowledge generated by this project will help establish a general framework for understanding the role of PTMs in gene regulation, which is highly anticipated but still sorely missing. This knowledge will cement the broader impact of this project by shedding new light onto the functions of the epitranscriptome in many other processes involving RNA, which will help increase our understanding of the myriad health conditions linked to RNA malfunction.